An alteration in vascular permeability is a defining feature of diverse processes including arteriosclerosis, inflammation, acute lung injury (ALI) and angiogenesis. In contrast, there is little known about processes that determine barrier protection or barrier restoration after edemagenic agents. We have previously shown that hepatocyte growth factor (HGF) binding to its cell surface receptor tyrosine kinase, c-Met, promotes increased EC barrier function via cytoskeletal rearrangement and attenuates inflammatory lung edema formation. Our data indicate that HGF promotes c-Met recruitment into specialized caveolin-1-enriched plasma membrane microdomains known as lipid rafts and transactivates sphingosine 1-phosphate receptor (S1P1) and CD44 (a major hyaluronan glycoprotein receptor) within these lipid raft structures. We have identified several potential regulators of HGF/c-Met-induced actin cytoskeletal rearrangement and consequent EC barrier enhancement in lipid rafts including the Rac1 exchange factor, Tiaml, the vesicle regulatory protein, dynamin 2, myosin light chain kinase (MLCK) and paxillin. Further, genes encoding c-Met and paxillin contain coding single nucleotide polymorphisms (SNPs) which potentially alter function and lead to increased susceptibility to ALI. Specific Aim #1 will identify the role of these SNPs in HGF/c-Met signaling from lipid rafts to the actin cytoskeleton and consequent human EC barrier regulation. Our preliminary data in murine model of lipopolysaccharide (LPS)-induced pulmonary vascular hyper-permeability suggests that caveolin-1 regulates HGF-mediated vascular integrity in vivo. Specific Aim #2 will define HGF/c-Met/caveolin-1 interactions in the regulation of endothelial cortical actin formation, tension and lung permeability. Further, our published data indicates that HGF/c-Met transactivation of the S1P1 is crucial for its EC barrierenhancing properties. Thus, to explore growth factor transactivation in regulating EC barrier function, Specific Aim #3 will identify HGF/c-Met/S1P1 interactions in the regulation of endothelial cortical actin formation, tension and lung permeability. Specific Aim #4 will define HGF/c-Met/CD44 interactions in the regulation of endothelial cortical actin formation, tension and lung permeability. Increased understanding of HGF/c-Met-mediated signal transduction and EC barrier regulation from lipid rafts may provide novel therapies for a variety of disease processes involving defects in EC barrier regulation.